Methods for blocking an interaction between notch-1 and delta-like 4 (DLL-4) by administering a DDL-4 antibody

ABSTRACT

There is disclosed compositions and methods relating to or derived from anti-DLL-4 antibodies. More specifically, there is disclosed fully human antibodies that bind DLL-4, DLL-4-binding fragments and derivatives of such antibodies, and DLL-4-binding polypeptides comprising such fragments. Further still, there is disclosed nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having DLL-4 related disorders or conditions, including various inflammatory disorders and various cancers.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a divisional of U.S. Ser. No. 13/903,793,filed May 28, 2013, which claims priority to U.S. provisional patentapplication 61/654,019 filed 31 May 2012.

TECHNICAL FIELD

The present disclosure provides compositions and methods relating to orderived from anti-DLL-4 antibodies. More specifically, the presentdisclosure provides human antibodies that bind DLL-4, DLL-4-bindingfragments and derivatives of such antibodies, and DLL-4-bindingpolypeptides comprising such fragments. Further still, the presentdisclosure provides antibodies, antibody fragments and derivatives andpolypeptides, and methods of using such antibodies, antibody fragmentsand derivatives and polypeptides, including methods of treating ordiagnosing subjects having DLL-4-related disorders or conditions.

BACKGROUND

Cell-to-cell communication is required for many biological processessuch as differentiation, proliferation, and homeostasis. One systemutilized by a wide range of eukaryotes is the Notch-signaling pathway.This pathway, especially the Notch receptor, is also critical forfunctional tumor angiogenesis. Thus, inhibition of Notch receptorfunction, blockage of the Notch receptor, and/or blockage of theNotch-signaling pathway are potential strategies for anti-cancercompositions and therapies. Small molecule inhibitors of the Notchreceptor have proven to be toxic because they suppress wild type(normal) tissue expression of Notch receptors throughout the body. Thus,different members of the Notch-signaling pathway should be considered aspotential targets fortherapeutics.

A vasculature ligand for the Notch receptor is Delta 4 or Delta-like 4(DLL-4). Largely expressed in the vasculature, DLL-4 is critical forvascular development (Yan et al., Clin. Cancer Res., 13(24): 7243-7246(2007); Shutter et al., Genes Dev., 14(11): 1313-1318 (2000); Gale etal., Proc. Natl. Acad. Sci. USA. 101(45): 15949-15954 (2004); Krebs etal., Genes Dev., 14(11): 1343-1352 (2000)). Mice heterozygous for DLL-4are embryonically lethal due to major defects in vascular development(Gale et al., Proc. Natl. Acad. Sci. USA, 101(45): 15949-15954 (2004);Duarte et al., Genes Dev., 18(20): 2474-2478 (2004); Krebs et al., GenesDev., 18(20): 2469-2473 (2004)). The expression of DLL-4 can be inducedby VEGF (Liu et al., Mal. Cell. Biol., 23(1): 14-25 (2003); Lobov etal., Proc. Natl. Acad. Sci. USA, 104(9): 3219-3224 (2007)). In sum,DLL-4 can negatively regulate VEGF signaling, in part through repressingVEGFR2 and inducing VEGFR1 (Harrington et al., Microvasc. Res., 75(2):144-154 (2008); Suchting et al., Proc. Natl. Acad. Sci. USA, 104(9):3225-3230 (2007)). Exquisite coordination between DLL4 and VEGF isessential for functional angiogenesis.

In addition to its physiological role. DLL-4 is up-regulated in tumorblood vessels (Gale et al., Proc. Natl. Acad. Sci. USA, 101(45):15949-15954 (2004); Mailhos et al., Differentiation, 69(2-3): 135-144(2001); Patel et al., Cancer Res., 65(19): 8690-8697 (2005); Patel etal., Clin. Cancer Res., 12(16): 4836-4844 (2006); Noguera-Troise et al.,Nature, 444(7122): 1032-1037 (2006)). Blockade of DLL-4 potentlyinhibited primary tumor growth in multiple models (Noguera-Troise etal., Nature, 444(7122): 1032-1037 (2006); Ridgway et al., Nature,444(7122): 1083-1087 (2006); Scehnet et al., Blood, 109(11): 4753-4760(2007)). The inhibition of DLL-4 was even effective against tumors thatare resistant to anti-VEGF therapy. The combinatorial inhibition of bothDLL-4 and VEGF provided an enhanced anti-tumor activity. Interestingly,unlike VEGF inhibition that reduces tumor vessel formation, DLL-4blockade leads to an increase in tumor vasculature density wherein thevessels are abnormal, cannot support efficient blood transport, and areeffectively nonfunctional. Thus, DLL4 provides a potential target forcancer treatment.

Interactions between Notch receptors and their ligands represent anevolutionarily conserved pathway important not only for cell fatedecisions but also in regulating lineage decisions in hematopoiesis andin the developing thymus (Artavanis-Tsakonas et al. 1999, Science284:770-776; Skokos et al. 2007; J. Exp. Med. 204:1525-1531; and Amsenet al. 2004, Cell 117:515-526). It has been recently shown thatDLL-4-Notch 1 inhibition leads to a complete block in T cell developmentaccompanied by ectopic appearance of B cells and an expansion ofdendritic cells (DC) that can arise from Pro-T cell to DC fateconversion within the thymus (Hozumi et al. 2008, J. Exp. Med.205(11):2507-2513; Koch et al. 2008, J. Exp. Med. 205(11):2515-2523; andFeyerabend et al. 2009. Immunity 30:1-13). Thus, there is accumulatingevidence that Notch signaling is critical for the determination of cellfate decision from hematopoietic progenitor cells. Furthermore, afeedback control of regulatory T cell (Treg) homeostasis by DCs in vivohas been shown (Darrasse-Jeze et al. 2009, J. Exp. Med. 206(9):1853-1862). However, the role of Notch signaling in controlling theorigin and the development of DCs and consequently Treg homeostasis isstill unknown. This is a question that is clinically important becauseidentifying new methods of inducing Treg expansion could be used as atreatment for autoimmunity diseases and disorders.

Other DLL antagonists and their uses are disclosed in WO 2007/143689, WO2007/070671, WO 2008/076379, WO 2008/042236, and WO/2008/019144.Therefore, there is a need in the art for therapeutic agents capable oftargeting the DLL-4-Notch pathway and thereby inhibiting, or evenpreventing, tumor angiogenesis and growth.

SUMMARY

The present disclosure provides a fully human antibody of an IgG classthat binds to a DLL-4 epitope with a binding affinity of at least 10⁻⁶M, which has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQID NO. 39, and combinations thereof, and that has a light chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ IDNO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, and combinationsthereof. Preferably, the fully human antibody has both a heavy chain anda light chain wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 (called C3 herein), SEQ ID NO. 3/SEQ ID NO. 4 (calledC5 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called C10 herein), SEQ ID NO.7/SEQ ID NO. 8 (called G6 herein), SEQ ID NO. 9/SEQ ID NO. 10 (calledF11 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called D2 herein), SEQ ID NO.13/SEQ ID NO. 14 (called E10 herein), SEQ ID NO. 15/SEQ ID NO. 16(called F12 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called E5 herein), SEQID NO. 19/SEQ ID NO. 20 (called H2 herein), SEQ ID NO. 21/SEQ ID NO. 22(called A1 herein). SEQ ID NO. 23/SEQ ID NO. 24 (called A2 herein), SEQID NO. 25/SEQ ID NO. 26 (called A11 herein), SEQ ID NO. 27/SEQ ID NO. 28(called C12 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called D9 herein). SEQID NO. 31/SEQ ID NO. 32 (called E3 herein), SEQ ID NO. 33/SEQ ID NO. 34(called E6 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called F1 herein), SEQID NO. 37/SEQ ID NO. 38 (called F6 herein). SEQ ID NO. 39/SEQ ID NO. 40(called F10 herein), and combinations thereof.

The present disclosure provides a Fab fully human antibody fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinations thereof,and that has a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2. SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38,SEQ ID NO. 40, and combinations thereof. Preferably, the fully humanantibody Fab fragment has both a heavy chain variable domain region anda light chain variable domain region wherein the antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQID NO. 5/SEQ ID NO. 6. SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ IDNO. 10. SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ IDNO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18. SEQ ID NO. 19/SEQ IDNO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ IDNO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ IDNO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ IDNO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ IDNO. 40, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21. SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, and combinations thereof. Preferably, thefully human single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8. SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22. SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32. SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, comprising administering an effectiveamount of an anti-DLL-4 polypeptide, wherein the anti-DLL-4 polypeptideis selected from the group consisting of a fully human antibody of anIgG class that binds to a DLL-4 epitope with a binding affinity of atleast 10⁻⁶ M, a Fab fully human antibody fragment, having a variabledomain region from a heavy chain and a variable domain region from alight chain, a single chain human antibody, having a variable domainregion from a heavy chain and a variable domain region from a lightchain and a peptide linker connection the heavy chain and light chainvariable domain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5. SEQ ID NO. 7. SEQ ID NO. 9, SEQ ID NO. 11. SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25. SEQ ID NO. 27. SEQ ID NO. 29. SEQ ID NO. 31. SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinations thereof,and that has a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4. SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38,SEQ ID NO. 40, and combinations thereof;

wherein the Fab fully human antibody fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1. SEQID NO. 3. SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23. SEQ ID NO. 25. SEQ ID NO. 27. SEQ ID NO. 29. SEQ ID NO.31. SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, andcombinations thereof, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, and combinations thereof;and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinationsthereof, and that has the light chain variable domain sequence that isat least 95% identical to the amino acid sequences selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.8, SEQ ID NO. 10. SEQ ID NO. 12. SEQ ID NO. 14. SEQ ID NO. 16, SEQ IDNO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36,SEQ ID NO. 38, SEQ ID NO. 40, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called C3 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called C5 herein), SEQ IDNO. 5/SEQ ID NO. 6 (called C10 herein), SEQ ID NO. 7/SEQ ID NO. 8(called G6 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called F11 herein), SEQID NO. 11/SEQ ID NO. 12 (called D2 herein), SEQ ID NO. 13/SEQ ID NO. 14(called E10 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called F12 herein),SEQ ID NO. 17/SEQ ID NO. 18 (called E5 herein), SEQ ID NO. 19/SEQ ID NO.20 (called H2 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called A1 herein),SEQ ID NO. 23/SEQ ID NO. 24 (called A2 herein), SEQ ID NO. 25/SEQ ID NO.26 (called A11 herein). SEQ ID NO. 27/SEQ ID NO. 28 (called C12 herein),SEQ ID NO. 29/SEQ ID NO. 30 (called D9 herein), SEQ ID NO. 31/SEQ ID NO.32 (called E3 herein), SEQ ID NO. 33/SEQ ID NO. 34 (called E6 herein),SEQ ID NO. 35/SEQ ID NO. 36 (called F1 herein). SEQ ID NO. 37/SEQ ID NO.38 (called F6 herein), SEQ ID NO. 39/SEQ ID NO. 40 (called F10 herein),and combinations thereof. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 (called C3 herein), SEQ ID NO. 3/SEQ ID NO. 4(called C5 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called C10 herein), SEQID NO. 7/SEQ ID NO. 8 (called G6 herein), SEQ ID NO. 9/SEQ ID NO. 10(called F11 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called D2 herein), SEQID NO. 13/SEQ ID NO. 14 (called E10 herein), SEQ ID NO. 15/SEQ ID NO. 16(called F12 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called E5 herein), SEQID NO. 19/SEQ ID NO. 20 (called H2 herein), SEQ ID NO. 21/SEQ ID NO. 22(called A1 herein), SEQ ID NO. 23/SEQ ID NO. 24 (called A2 herein), SEQID NO. 25/SEQ ID NO. 26 (called A11 herein), SEQ ID NO. 27/SEQ ID NO. 28(called C12 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called D9 herein), SEQID NO. 31/SEQ ID NO. 32 (called E3 herein), SEQ ID NO. 33/SEQ ID NO. 34(called E6 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called F1 herein), SEQID NO. 37/SEQ ID NO. 38 (called F6 herein), SEQ ID NO. 39/SEQ ID NO. 40(called F10 herein), and combinations thereof. Preferably, the fullyhuman single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. S/SEQ ID NO. 6. SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10. SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18. SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, and combinations thereof.

Preferably, the broad spectrum of mammalian cancers to be treated isselected from the group consisting of ovarian, colon, breast, lungcancers, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemias. T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, mast cell derived tumors, andcombinations thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the initial screening binding of various anti-DLL-4antibodies to cell surface expressed DLL-4. Cells stably expressingDLL-4 were incubated with 1 μg/ml of anti-DLL-4 antibodies as indicated.The binding was analyzed by flow cytometry (HTFC, Intellicyt).

FIG. 1B shows the binding of exemplary anti-DLL-4 antibodies to cellsurface expressed DLL-4. Cells stably expressing DLL-4 were incubatedwith increasing amounts of anti-DLL-4 antibodies. The binding wasanalyzed by flow cytometry. Anti-DLL-4 antibodies showed bindingcharacteristics (EC₅₀) comparable to or better than Oncomed OMP21M18.

FIG. 2 shows a Biacore analysis of various anti-DLL-4 antibodies. Theresulting binding kinetic parameters are indicated in the accompanyingtable.

FIGS. 3A and 3B show epitope mapping of anti-DLL-4 antibodies inrelation to Oncomed OMP21M18 antibody. Additional binding registered byOctet indicates that anti-DLL-4 antibodies C3, C5 or G6 binds to anepitope of DLL-4 that is not occupied by 21M18.

FIG. 4A shows how anti-DLL-4 antibodies block rhDLL4 binding toimmobilized rrNotch. The greater the binding the higher the absorbancevalue, therefore the best blocking antibodies are C10 and G6 which showthe lowest values and are comparable to Oncomed OMP21M18 on the basis ofin vitro binding activity.

FIG. 4B shows that anti-DLL-4 antibodies block the binding of a solublerecombinant Notch-1 (either rat or human) to cellular human DLL-4, asmeasured by flow cytometry. In this assay, Notch-1 is fluorescentlylabeled, therefore a high Mean Fluorescence Intensity (MFI) wouldreflect a strong interaction between Notch-1 and DLL-4. In the presenceof anti-DLL-4 antibodies, little to no fluorescence was detectedindicating blocking of the interaction.

FIG. 4C shows the dose-dependent inhibition of the DLL-4-Notch-1interaction for selected antibodies. IC₅₀ values reflect theconcentration of antibody which causes 50% inhibition of interactionbetween soluble Notch-1 and DLL-4. Anti-DLL-4 antibodies disclosedwithin show greater inhibitory activity than OMP21M18.

FIG. 5 shows a RT-PCR-based bioassay that was developed to determine theability of selected antibodies to neutralize DLL-4-mediated cellularfunction in vitro. Data show that anti-DLL-4 antibody C3, an exemplaryantibody of this disclosure, was able to efficiently inhibitDLL-4-mediated activation of Notch-1 dependent gene expression in abreast cancer cell Line (MCF7).

FIG. 6 shows that anti-DLL-4 antibodies can prevent DLL-4-mediated HUVECproliferation inhibition. DLL4 is known to inhibit VEGF-mediated HUVECproliferation. Thus, when cultured on DLL-4-coated plate, HUVEC growthis inhibited. Data show that anti-DLL-4 antibodies were able to blockthis inhibition and to partially restore HUVEC growth.

DETAILED DESCRIPTION

The present disclosure provides a fully human antibody of an IgG classthat binds to a DLL-4 epitope with a binding affinity of 10*M or less,that has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3. SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 23. SEQ ID NO. 25. SEQ ID NO. 27, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQID NO. 39, and combinations thereof, and that has a light chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ IDNO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, and combinationsthereof. Preferably, the fully human antibody has both a heavy chain anda light chain wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 (called C3 herein), SEQ ID NO. 3/SEQ ID NO. 4 (calledC5 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called C10 herein), SEQ ID NO.7/SEQ ID NO. 8 (called G6 herein). SEQ ID NO. 9/SEQ ID NO. 10 (called FI1 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called D2 herein), SEQ ID NO.13/SEQ ID NO. 14 (called E10 herein), SEQ ID NO. 15/SEQ ID NO. 16(called F12 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called E5 herein), SEQID NO. 19/SEQ ID NO. 20 (called H2 herein), SEQ ID NO. 21/SEQ ID NO. 22(called A1 herein). SEQ ID NO. 23/SEQ ID NO. 24 (called A2 herein), SEQID NO. 25/SEQ ID NO. 26 (called A11 herein), SEQ ID NO. 27/SEQ ID NO. 28(called C12 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called D9 herein). SEQID NO. 31/SEQ ID NO. 32 (called E3 herein), SEQ ID NO. 33/SEQ ID NO. 34(called E6 herein), SEQ ID NO. 35/SEQ ID NO. 36 (called F1 herein), SEQID NO. 37/SEQ ID NO. 38 (called F6 herein), SEQ ID NO. 39/SEQ ID NO. 40(called F10 herein), and combinations thereof.

The present disclosure provides a Fab fully human antibody fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31. SEQ ID NO. 33.SEQ ID NO. 35. SEQ ID NO. 37. SEQ ID NO. 39, and combinations thereof,and that has a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38,SEQ ID NO. 40, and combinations thereof. Preferably, the fully humanantibody Fab fragment has both a heavy chain variable domain region anda light chain variable domain region wherein the antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2. SEQ ID NO. 3/SEQ ID NO. 4, SEQID NO. 5/SEQ ID NO. 6. SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ IDNO. 10, SEQ ID NO. 11/SEQ ID NO. 12. SEQ ID NO. 13/SEQ ID NO. 14, SEQ IDNO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ IDNO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ IDNO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ IDNO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ IDNO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ IDNO. 40, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2. SEQ ID NO. 4. SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24. SEQ ID NO. 26. SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQID NO. 38, SEQ ID NO. 40, and combinations thereof. Preferably, thefully human single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4. SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8. SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28. SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, comprising administering an effectiveamount of an anti-DLL-4 polypeptide, wherein the anti-DLL-4 polypeptideis selected from the group consisting of a fully human antibody of anIgG class that binds to a DLL-4 epitope with a binding affinity of atleast 10⁻⁶M, a Fab fully human antibody fragment, having a variabledomain region from a heavy chain and a variable domain region from alight chain, a single chain human antibody, having a variable domainregion from a heavy chain and a variable domain region from a lightchain and a peptide linker connection the heavy chain and light chainvariable domain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5. SEQ ID NO. 7. SEQ ID NO. 9, SEQ ID NO. 11. SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinations thereof,and that has a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28. SEQID NO. 30. SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38,SEQ ID NO. 40, and combinations thereof;

wherein the Fab fully human antibody fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3. SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11. SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, andcombinations thereof, and that has the light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32. SEQ ID NO. 34.SEQ ID NO. 36. SEQ ID NO. 38. SEQ ID NO. 40, and combinations thereof;and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29. SEQ ID NO. 31. SEQID NO. 33. SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, and combinationsthereof, and that has the light chain variable domain sequence that isat least 95% identical to the amino acid sequences selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.8, SEQ ID NO. 10. SEQ ID NO. 12. SEQ ID NO. 14. SEQ ID NO. 16, SEQ IDNO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36,SEQ ID NO. 38, SEQ ID NO. 40, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4. SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8. SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16. SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28. SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, and combinations thereof.Preferably, the fully human antibody Fab fragment has both a heavy chainvariable domain region and a light chain variable domain region whereinthe antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ IDNO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO.8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12. SEQ ID NO.13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO.23/SEQ ID NO. 24. SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO.28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO.38. SEQ ID NO. 39/SEQ ID NO. 40, and combinations thereof. Preferably,the fully human single chain antibody has both a heavy chain variabledomain region and a light chain variable domain region, wherein thesingle chain fully human antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ IDNO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO.12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18. SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28. SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 33/SEQ ID NO. 34, SEQ ID NO. 35/SEQ ID NO. 36, SEQ ID NO.37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, and combinations thereof.

Preferably, the broad spectrum of mammalian cancers to be treated isselected from the group consisting of ovarian, colon, breast, lungcancers, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemias, T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, mast cell derived tumors, andcombinations thereof.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example.Korndorfer et al., 2003, Proteins: Structure. Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronectin components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT® (international ImMunoGeneTics information system; Lefrancet al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger andPluckthun, J. Mol. Biol. 309(3):657-670; 2001).

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies usually are made of less than about 10% antibody havingspecific binding activity for the particular antigen. Subjecting thesepreparations to several rounds of affinity purification can increase theproportion of antibody having specific binding activity for the antigen.Antibodies prepared in this manner are often referred to as“monospecific.” Monospecfic antibody preparations can be made up ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99%, or 99.9% antibody having specific binding activity for theparticular antigen.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L),and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US App. Pub.20/0202512; 2004/0202995; 2004/0038291; 2004/0009507; 2003/0039958, andWard et al., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (Bird et al., 1988. Science242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (Holliger etal., 1993. Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al.,1994, Structure 2:1121-23). If the two polypeptide chains of a diabodyare identical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. supra: Lefranc et al., supra and/or Honegger and Pluckthun,supra. One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an antigen binding protein. Anantigen binding protein may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-DLL-4 antibody. In another embodiment, all of the CDRsare derived from a human anti-DLL-4 antibody. In another embodiment, theCDRs from more than one human anti-DLL-4 antibodies are mixed andmatched in a chimeric antibody. For instance, a chimeric antibody maycomprise a CDR1 from the light chain of a first human anti-PAR-2antibody, a CDR2 and a CDR3 from the light chain of a second humananti-DLL-4 antibody, and the CDRs from the heavy chain from a thirdanti-DLL-4 antibody. Other combinations are possible.

Further, the framework regions may be derived from one of the sameanti-DLL-4 antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind DLL-4).

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the proteolytic activation of DLL-4 when an excess of theanti-DLL-4 antibody reduces the amount of activation by at least about20% using an assay such as those described herein in the Examples. Invarious embodiments, the antigen binding protein reduces the amount ofamount of proteolytic activation of DLL-4 by at least 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. (Bowie et al., 1991,Science 253:164).

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human DLL-4) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego. Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamsterovary (CHO) cells or their derivatives such as Veggie CHO and relatedcell lines which grow in serum-free media (Rasmussen et al., 1998,Cytotechnology 28:31) or CHO strain DX-BI 1, which is deficient in DHFR(Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLacells. BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) (McMahan etal., 1991, EMBO J. 10:2821), human embryonic kidney cells such as293,293 EBNA or MSR 293, human epidermal A431 cells, human Colo205cells, other transformed primate cell lines, normal diploid cells, cellstrains derived from in vitro culture of primary tissue, primaryexplants, HL-60, U937, HaK or Jurkat cells. Typically, a host cell is acultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Preferably, the mammalian cancer to be treated is selected from thegroup consisting of ovarian, colon, breast or hepatic carcinoma celllines, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemia's, T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, mast cell derived tumors, andcombinations thereof.

Polypeptides of the present disclosure can be produced using anystandard methods known in the art. In one example, the polypeptides areproduced by recombinant DNA methods by inserting a nucleic acid sequence(e.g., a cDNA) encoding the polypeptide into a recombinant expressionvector and expressing the DNA sequence under conditions promotingexpression. Examples of nucleic acid sequences encoding VK-8Bpolypeptide disclosed herein are:

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. (Mayfield et al., Proc. Natl. Acad. Sci.USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002(1):96-105; Connell, Curr. Opin. Biotechnol. 2001 12(5):446-9; Makrideset al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 19917(7):657-78).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or Ausubelet al., Current Protocols in Molecular Biology (Green Publishing andWiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. Moreover, a host can replicate, usuallyconferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

DLL-4-binding polypeptides can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. (Raju et al. Biochemistry. 2001 31; 40(30):8868-76). Effects ofsuch non-amino acid elements on the functionality of a polypeptide maybe tested for its antagonizing role in DLL-4 function, e.g., itsinhibitory effect on angiogenesis or on tumor growth.

In one specific embodiment, modified forms of the subject solublepolypeptides comprise linking the subject soluble polypeptides tononproteinaceous polymers. In one specific embodiment, the polymer ispolyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes,in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4.301.144; 4,670,417; 4,791,192 or 4,179,337.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York. Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)-1CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

Although PEG is well-known, this is, to our knowledge, the firstdemonstration that a pegylated^(10F)n3 polypeptide can be pegylated andretain ligand binding activity. In a preferred embodiment, thepegylated^(10F)n3 polypeptide is produced by site-directed pegylation,particularly by conjugation of PEG to a cysteine moiety at the N- orC-terminus. Accordingly, the present disclosure provides atarget-binding ^(10F)n3 polypeptide with improved pharmacokineticproperties, the polypeptide comprising: a ^(10F)n3 domain having fromabout 80 to about 150 amino acids, wherein at least one of the loops ofsaid ^(10F)n3 domain participate in target binding; and a covalentlybound PEG moiety, wherein said ^(10F)n3 polypeptide binds to the targetwith a K_(D) of less than 100 nM and has a clearance rate of less than30 mL/hr/kg in a mammal. The PEG moiety may be attached to the ^(10F)n3polypeptide by site directed pegylation, such as by attachment to a Cysresidue, where the Cys residue may be positioned at the N-terminus ofthe ^(0F)n3 polypeptide or between the N-terminus and the mostN-terminal beta or beta-like strand or at the C-terminus of the ^(10F)n3polypeptide or between the C-terminus and the most C-terminal beta orbeta-like strand. A Cys residue may be situated at other positions aswell, particularly any of the loops that do not participate in targetbinding. A PEG moiety may also be attached by other chemistry, includingby conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem.,252, 3582 (1977). Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to DLL-4-binding polypeptides. The number of repeating units“n” in the PEG is approximated for the molecular mass described inDaltons. It is preferred that the combined molecular mass of PEG on anactivated linker is suitable for pharmaceutical use. Thus, in oneembodiment, the molecular mass of the PEG molecules does not exceed100,000 Da. For example, if three PEG molecules are attached to alinker, where each PEG molecule has the same molecular mass of 12.000 Da(each n is about 270), then the total molecular mass of PEG on thelinker is about 36,000 Da (total n is about 820). The molecular massesof the PEG attached to the linker can also be different, e.g., of threemolecules on a linker two PEG molecules can be 5,000 Da each (each n isabout 110) and one PEG molecule can be 12,000 Da (n is about 270).

In a specific embodiment a DLL-4 binding polypeptide is covalentlylinked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a binding polypeptide's6-amino group of a lysine is the available (free) amino group.

The above conjugates may be more specifically presented by formula (II):P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR (II), wherein P is the group of abinding polypeptide as described herein, (i.e. without the amino groupor amino groups which form an amide linkage with the carbonyl shown informula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about450 to about 950 and is chosen so that the molecular weight of theconjugate minus the binding polypeptide is from about 10 to about 40kDa. As used herein, the given ranges of “m” have an orientationalmeaning. The ranges of “m” are determined in any case, and exactly, bythe molecular weight of the PEG group. One skilled in the art can selecta suitable molecular mass for PEG, e.g., based on how the pegylatedbinding polypeptide will be used therapeutically, the desired dosage,circulation time, resistance to proteolysis, immunogenicity, and otherconsiderations.

In one embodiment, PEG molecules may be activated to react with aminogroups on a binding polypeptide, such as with lysines (Bencham et al.,Anal. Biochem., 131, 25 (1983); Veronese et al., Appl. Biochem., 11, 141(1985).; Zalipsky et al., Polymeric Drugs and Drug Delivery Systems,adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky et al., Europ.Polym. J., 19, 1177-1183 (1983); Delgado et al., Biotechnology andApplied Biochemistry, 12, 119-128 (1990)).

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similar typeof reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of a ^(10F)n3 polypeptide can be performed according to themethods of the state of the art, for example by reaction of the bindingpolypeptide with electrophilically active PEGs (supplier. ShearwaterCorp., USA, www.shearwatercorp.com). Preferred PEG reagents of thepresent invention are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini et al., BioconjugateChem. 6 (1995) 62-69). Such methods may be used to pegylate at anf-amino group of a binding polypeptide lysine or the N-terminal aminogroup of the binding polypeptide.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a binding polypeptide (Sartore et al., Appl. Biochem. Biotechnol.,27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368 (1996);Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897).U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplary reactive PEGspecies that may be coupled to sulfhydryl groups.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a binding polypeptide, the cysteine residues are native tothe binding polypeptide, whereas in other embodiments, one or morecysteine residues are engineered into the binding polypeptide. Mutationsmay be introduced into a binding polypeptide coding sequence to generatecysteine residues. This might be achieved, for example, by mutating oneor more amino acid residues to cysteine. Preferred amino acids formutating to a cysteine residue include serine, threonine, alanine andother hydrophilic residues. Preferably, the residue to be mutated tocysteine is a surface-exposed residue. Algorithms are well-known in theart for predicting surface accessibility of residues based on primarysequence or a protein. Alternatively, surface residues may be predictedby comparing the amino acid sequences of binding polypeptides, giventhat the crystal structure of the framework based on which bindingpolypeptides are designed and evolved has been solved (Himanen et al.,Nature. (2001) 20-27; 414(6866):933-8) and thus the surface-exposedresidues identified. In one embodiment, cysteine residues are introducedinto binding polypeptides at or near the N- and/or C-terminus, or withinloop regions.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297, 1059, and U.S. Pat. No. 5,824,784.The use of a PEG-aldehyde for the reductive amination of a proteinutilizing other available nucleophilic amino groups is described in U.S.Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579,and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

The ratio of a binding polypeptide to activated PEG in the conjugationreaction can be from about 1:0.5 to 1:50, between from about 1:1 to1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used inthe present method to catalyze the covalent addition of PEG to thebinding polypeptide. In one embodiment, the pH of a buffer used is fromabout 7.0 to 9.0. In another embodiment, the pH is in a slightly basicrange, e.g., from about 7.5 to 8.5. Buffers having a pKa close toneutral pH range may be used, e.g., phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g. gel filtration) and ion exchange chromatography. Products may alsobe separated using SDS-PAGE. Products that may be separated includemono-, di-, tri- poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, PEGylated binding polypeptide of the inventioncontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin PEG-binding polypeptide is from about 3,000 Da to 60,000 Da,optionally from about 10,000 Da to 36,000 Da.

In one embodiment of the invention, the PEG in pegylated bindingpolypeptide is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-bindingpolypeptide, more preferably at least 90%, and most preferably at least95%.

In another embodiment, the pegylated binding polypeptides of theinvention will preferably retain at least 25%, 50%, 60%, 70%, 80%, 85%,90%, 95% or 100% of the biological activity associated with theunmodified protein. In one embodiment, biological activity refers to itsability to bind to DLL-4, as assessed by KD, k_(on), or k_(off). In onespecific embodiment, the pegylated binding polypeptide protein shows anincrease in binding to DLL-4 relative to unpegylated bindingpolypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf-life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Formulations and Modes of Administration

The present disclosure features methods for treating conditions orpreventing pre-conditions which respond to an inhibition of DLL-4biological activity. Preferred examples are conditions that arecharacterized by inflammation or cellular hyperproliferation. Techniquesand dosages for administration vary depending on the type of specificpolypeptide and the specific condition being treated but can be readilydetermined by the skilled artisan. In general, regulatory agenciesrequire that a protein reagent to be used as a therapeutic is formulatedso as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

Exemplary Uses

The DLL-4 binding proteins described herein and their related variantsare useful in a number of therapeutic and diagnostic applications. Theseinclude the inhibition of the biological activity of DLL-4 by competingfor or blocking the binding to a DLL-4 as well as the delivery ofcytotoxic or imaging moieties to cells, preferably cells expressingDLL-4. The small size and stable structure of these molecules can beparticularly valuable with respect to manufacturing of the drug, rapidclearance from the body for certain applications where rapid clearanceis desired or formulation into novel delivery systems that are suitableor improved using a molecule with such characteristics.

On the basis of their efficacy as inhibitors of DLL-4 biologicalactivity, the polypeptides of this disclosure are effective against anumber of cancer conditions as well as complications arising fromcancer, such as pleural effusion and ascites. Preferably, theDLL-4-binding polypeptides of the disclosure can be used for thetreatment of prevention of hyperproliferative diseases or cancer and themetastatic spread of cancers. Preferred indications for the disclosedanti-DLL-4 antibodies include colorectal cancers, head and neck cancers,small cell lung cancer, non-small cell lung cancer (NSCLC) andpancreatic cancer. Non-limiting examples of cancers include bladder,blood, bone, brain, breast, cartilage, colon kidney, liver, lung, lymphnode, nervous tissue, ovary, pancreatic, prostate, skeletal muscle,skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,urogenital tract, ureter, urethra, uterus, or vaginal cancer.

In addition, various inflammatory disorders can be treated with thedisclosed anti-DLL-4 binding polypeptides disclosed herein. Suchinflammatory disorders include, for example, intestinal mucosainflammation wasting diseases associated with colitis, multiplesclerosis, rheumatoid arthritis, osteoarthritis, psoriasis, and Crohn'sdisease.

A DLL-4 binding polypeptide can be administered alone or in combinationwith one or more additional therapies such as chemotherapy radiotherapy,immunotherapy, surgical intervention, or any combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject anti-DLL-4 antibodies agents of theinvention can be used alone. Alternatively, the subject agents may beused in combination with other conventional anti-cancer therapeuticapproaches directed to treatment or prevention of proliferativedisorders (e.g., tumor). For example, such methods can be used inprophylactic cancer prevention, prevention of cancer recurrence andmetastases after surgery, and as an adjuvant of other conventionalcancer therapy. The present disclosure recognizes that the effectivenessof conventional cancer therapies (e.g., chemotherapy, radiation therapy,phototherapy, immunotherapy, and surgery) can be enhanced through theuse of a subject polypeptide therapeutic agent.

A wide array of conventional compounds have been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent maybe found to enhance the therapeutic effect of the anti-neoplastic agentor overcome cellular resistance to such anti-neoplastic agent. Thisallows decrease of dosage of an anti-neoplastic agent, thereby reducingthe undesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

Certain chemotherapeutic anti-tumor compounds may be categorized bytheir mechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (e.g., VEGF inhibitors, fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

Depending on the nature of the combinatory therapy, administration ofthe polypeptide therapeutic agents may be continued while the othertherapy is being administered and/or thereafter. Administration of thepolypeptide therapeutic agents may be made in a single dose, or inmultiple doses. In some instances, administration of the polypeptidetherapeutic agents is commenced at least several days prior to theconventional therapy, while in other instances, administration is beguneither immediately before or at the time of the administration of theconventional therapy.

The DLL-4 binding proteins described herein can also be detectablylabeled and used to contact cells expressing DLL-4 for imagingapplications or diagnostic applications. For diagnostic purposes, thepolypeptide of the invention is preferably immobilized on a solidsupport. Preferred solid supports include columns (for example, affinitycolumns, such as agarose-based affinity columns), microchips, or beads.

In one example of a diagnostic application, a biological sample, such asserum or a tissue biopsy, from a patient suspected of having a conditioncharacterized by inappropriate angiogenesis is contacted with adetectably labeled polypeptide of the disclosure to detect levels ofDLL-4. The levels of DLL-4 detected are then compared to levels of DLL-4detected in a normal sample also contacted with the labeled polypeptide.An increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%in the levels of the DLL-4 may be considered a diagnostic indicator.

In certain embodiments, the DLL-4 binding polypeptides are furtherattached to a label that is able to be detected (e.g., the label can bea radioisotope, fluorescent compound, enzyme or enzyme co-factor). Theactive moiety may be a radioactive agent, such as: radioactive heavymetals such as iron chelates, radioactive chelates of gadolinium ormanganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁴I, ¹³¹I, ¹³²I, or⁹⁹Tc. A binding agent affixed to such a moiety may be used as an imagingagent and is administered in an amount effective for diagnostic use in amammal such as a human and the localization and accumulation of theimaging agent is then detected. The localization and accumulation of theimaging agent may be detected by radioscintigraphy, nuclear magneticresonance imaging, computed tomography or positron emission tomography.Immunoscintigraphy using DLL-4 binding polypeptides directed at DLL-4may be used to detect and/or diagnose cancers and vasculature. Forexample, any of the binding polypeptide against a DLL-4 marker labeledwith ⁹⁹Technetium, ¹¹¹Indium, or ¹²⁵Iodine may be effectively used forsuch imaging. As will be evident to the skilled artisan, the amount ofradioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof the imaging agent to be administered based upon the specific activityand energy of a given radionuclide used as the active moiety. Typically0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries are administered. Thus,compositions according to the present invention useful as imaging agentscomprising a targeting moiety conjugated to a radioactive moietycomprise 0.1-100 millicuries, in some embodiments preferably 1-10millicuries, in some embodiments preferably 2-5 millicuries, in someembodiments more preferably 1-5 millicuries.

The DLL-4 binding polypeptides can also be used to deliver additionaltherapeutic agents (including but not limited to drug compounds,chemotherapeutic compounds, and radiotherapeutic compounds) to a cell ortissue expressing DLL-4. In one example, the DLL-4 binding polypeptideis fused to a chemotherapeutic agent for targeted delivery of thechemotherapeutic agent to a tumor cell or tissue expressing DLL-4.

The DLL-4 binding polypeptides are useful in a variety of applications,including research, diagnostic and therapeutic applications. Forinstance, they can be used to isolate and/or purify receptor or portionsthereof, and to study receptor structure (e.g., conformation) andfunction.

In certain aspects, the various binding polypeptides can be used todetect or measure the expression of DLL-4, for example, on endothelialcells (e.g., venous endothelial cells), or on cells transfected with aDLL-4 gene. Thus, they also have utility in applications such as cellsorting and imaging (e.g., flow cytometry, and fluorescence activatedcell sorting), for diagnostic or research purposes.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to DLL-4. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising a DLL-4protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the DLL-4 protein. In oneembodiment, a sample containing cells expressing a DLL-4 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing a DLL-4protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of a DLL-4protein in a biological sample can also be prepared. Such kits willinclude a DLL-4 binding polypeptide which binds to a DLL-4 protein orportion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present disclosure also provides a method of detectingand/or quantitating expression of DLL-4, wherein a compositioncomprising a cell or fraction thereof (e.g., membrane fraction) iscontacted with a binding polypeptide which binds to a DLL-4 or portionof the receptor under conditions appropriate for binding thereto, andthe binding is monitored. Detection of the binding polypeptide,indicative of the formation of a complex between binding polypeptide andDLL-4 or a portion thereof, indicates the presence of the receptor.Binding of a polypeptide to the cell can be determined by standardmethods, such as those described in the working examples. The method canbe used to detect expression of DLL-4 on cells from an individual.Optionally, a quantitative expression of DLL-4 on the surface ofendothelial cells can be evaluated, for instance, by flow cytometry, andthe staining intensity can be correlated with disease susceptibility,progression or risk.

The present disclosure also provides a method of detecting thesusceptibility of a mammal to certain diseases. To illustrate, themethod can be used to detect the susceptibility of a mammal to diseaseswhich progress based on the amount of DLL-4 present on cells and/or thenumber of DLL-4-positive cells in a mammal. In one embodiment, theinvention relates to a method of detecting susceptibility of a mammal toa tumor. In this embodiment, a sample to be tested is contacted with abinding polypeptide which binds to a DLL-4 or portion thereof underconditions appropriate for binding thereto, wherein the sample comprisescells which express DLL-4 in normal individuals. The binding and/oramount of binding is detected, which indicates the susceptibility of theindividual to a tumor, wherein higher levels of receptor correlate withincreased susceptibility of the individual to a tumor.

Polypeptide sequences are indicated using standard one- or three-letterabbreviations. Unless otherwise indicated, each polypeptide sequence hasamino termini at the left and a carboxy termini at the right; eachsingle-stranded nucleic acid sequence, and the top strand of eachdouble-stranded nucleic acid sequence, has a 5′ termini at the left anda 3′ termini at the right. A particular polypeptide sequence also can bedescribed by explaining how it differs from a reference sequence.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The terms “DLL-4 inhibitor” and “DLL-4 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of DLL-4. Conversely, a “DLL-4 agonist” is a molecule thatdetectably increases at least one function of DLL-4. The inhibitioncaused by a DLL-4 inhibitor need not be complete so long as it isdetectable using an assay. Any assay of a function of DLL-4 can be used,examples of which are provided herein. Examples of functions of DLL-4that can be inhibited by a DLL-4 inhibitor, or increased by a DLL-4agonist, include cancer cell growth or apoptosis (programmed celldeath), and so on. Examples of types of DLL-4 inhibitors and DLL-4agonists include, but are not limited to, DLL-4 binding polypeptidessuch as antigen binding proteins (e.g., DLL-4 inhibiting antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example.Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronectin components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG. IgA, and IgE, respectively.Preferably, the anti-DLL-4 antibodies disclosed herein are characterizedby their variable domain region sequences in the heavy V_(H) and lightV_(L) amino acid sequences. The preferred antibody is A6 which is akappa IgG antibody. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)). The variable regions of eachlight/heavy chain pair form the antibody binding site such that anintact immunoglobulin has two binding sites.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human DLL-4) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain, “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent homology” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

A “host cell” is a cell that can be used to express a nucleic acid. Ahost cell can be a prokaryote, for example, E. coli, or it can be aeukaryote, for example, a single-celled eukaryote (e.g., a yeast orother fungus), a plant cell (e.g., a tobacco or tomato plant cell), ananimal cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell) or a hybridoma. Examples of hostcells include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175). L cells. C127 cells, 3T3 cells(ATCC CCL 163). Chinese hamster ovary (CHO) cells or their derivativessuch as Veggie CHO and related cell lines which grow in serum-free media(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11,which is deficient in DHFR (Urlaub et al., 1980, Proc. Natl. Acad. Sci.USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNAcell line derived from the African green monkey kidney cell line CV1(ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonickidney cells such as 293,293 EBNA or MSR 293, human epidermal A431cells, human Colo205 cells, other transformed primate cell lines, normaldiploid cells, cell strains derived from in vitro culture of primarytissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, ahost cell is a cultured cell that can be transformed or transfected witha polypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Antigen Binding Proteins

Antigen binding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, antibody muteins, and antibody variants) are polypeptidesthat bind to DLL-4, (preferably, human DLL-4). Antigen binding proteinsinclude antigen binding proteins that inhibit a biological activity ofDLL-4.

Oligomers that contain one or more antigen binding proteins may beemployed as DLL-4 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have DLL-4 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990. Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a DLL-4 binding fragment of an anti-DLL-4 antibody tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-DLL-4 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-DLL-4 antibody fragments or derivatives thatform are recovered from the culture supernatant.

The present disclosure provides a DLL-4 binding antigen binding protein(for example, an anti-DLL-4 antibody), that has one or more of thefollowing characteristics: binds to both human and murine DLL-4,inhibits the activation of human DLL-4, inhibits the activation ofmurine DLL-4, binds to or near the proteolytic cleavage site of DLL-4,causes relatively little down-regulation of cell-surface expressedDLL-4.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments.

The present disclosure provides monoclonal antibodies that bind toDLL-4. Monoclonal antibodies may be produced using any technique knownin the art, e.g., by immortalizing spleen cells harvested from thetransgenic animal after completion of the immunization schedule. Thespleen cells can be immortalized using any technique known in the art,e.g., by fusing them with myeloma cells to produce hybridomas. Myelomacells for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

Antigen binding proteins directed against DLL-4 can be used, forexample, in assays to detect the presence of DLL-4 polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying DLL-4 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as DLL-4 antagonists may beemployed in treating any DLL-4-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit DLL-4-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic activation of DLL-4, examples of which are provided herein,thus may be treated. In one embodiment, the present invention provides atherapeutic method comprising in vivo administration of a DLL-4 blockingantigen binding protein to a mammal in need thereof in an amounteffective for reducing an DLL-4-induced biological activity.

Antigen binding proteins include fully human monoclonal antibodies thatinhibit a biological activity of DLL-4.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses. Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells. HeLa cells. BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC: CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of DLL-4bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-DLL-4 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-DLL-4 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP→CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for DLL-4 of at least 10⁶. Inother embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰, In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from DLL-4. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to ⁻¹ orlower. In another embodiment, the K_(off) is 5×10⁻⁵ to ⁻¹ or lower. Inanother embodiment, the K_(off) is substantially the same as an antibodydescribed herein. In another embodiment, the antigen binding proteinbinds to DLL-4 with substantially the same K_(off) as an antibodydescribed herein.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of DLL-4. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower, in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of DLL-4with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to human DLL-4 expressed on the surface of a celland, when so bound, inhibits DLL-4 signaling activity in the cellwithout causing a significant reduction in the amount of DLL-4 on thesurface of the cell. Any method for determining or estimating the amountof DLL-4 on the surface and/or in the interior of the cell can be used.In other embodiments, binding of the antigen binding protein to theDLL-4-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface DLL-4 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of DLL-4, or to anepitope of DLL-4 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise a DLL-4 binding site from oneof the herein-described antibodies and a second DLL-4 binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another DLL-4 antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold) bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Pharmaceutical compositions comprising the antibodies and fragmentsthereof of the disclosure are administered to a subject in a mannerappropriate to the indication. Pharmaceutical compositions may beadministered by any suitable technique, including but not limited to,parenterally, topically, or by inhalation. If injected, thepharmaceutical composition can be administered, for example, viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal or subcutaneous routes, by bolus injection, orcontinuous infusion. Localized administration, e.g. at a site of diseaseor injury is contemplated, as are transdermal delivery and sustainedrelease from implants. Delivery by inhalation includes, for example,nasal or oral inhalation, use of a nebulizer, inhalation of theantagonist in aerosol form, and the like. Other alternatives includeeyedrops; oral preparations including pills, syrups, lozenges or chewinggum; and topical preparations such as lotions, gels, sprays, andointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds DLL-4 ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to an DLL-4binding antigen binding protein.

Example 1

This example illustrates in vitro data for cell binding as part of aninitial screen for several disclosed antibodies. CHO-K1 cells stablyexpressing DLL-4 (CHO-DLL-4 cells) were lifted in enzyme-freeDissociation Buffer and transferred to V-Bottom 96 well-plates (100,000cells/well). Cells were incubated with anti-DLL-4 Antibodies in FACSbuffer (PBS+2% FBS)+NaN₃ on ice for 45 min. After 2 washes in FACSbuffer, a 1:1000 dilution of Phycoerythrin conjugated anti-Human IgG(γ-chain specific) was added and incubated for 30 min. Following a finalwash, fluorescence intensity was measured on an Intellicyt HighThroughput Flow Cytometer (HTFC). Data were analyzed using GraphpadPrism software using non-linear regression fit. Data points are shown asthe median fluorescence intensity (MFI) of positively labeled cells +/−Standard Error. EC₅₀ values are reported as the concentration ofantibody to achieve 50% of maximal DLL-4 antibodies binding to DLL-4expressing cells.

Results: This example shows that the disclosed anti-DLL-4 monoclonalantibodies were able to bind efficiently to DLL-4 expressed on thesurface of cells. Several disclosed antibodies showed more potentbinding characteristics compared to anti-DLL-4 antibody OMP21M18(Oncomed), currently in clinical trial. EC₅₀ values were calculated forthe disclosed anti-DLL4 antibodies C3, C10 and G6, and range from 0.1 to0.3 nM, compared to 1.7 nM for OMP21M18 (FIG. 2).

Example 2

This example illustrates an antigen binding affinity determination fordisclosed anti-DLL4 antibodies C3, C6 and G6. Affinity DeterminationUsing BIACORE® Surface Plasmon Resonance Technology. Recombinant hDLL4was immobilized on CM5 sensor chip using standard NHS/EDC couplingmethodology. All measurements were conducted in HBS-EP buffer with aflow rate of 30 μL/min. The antibody was diluted so as to obtain aseries of concentrations, a 1:1 (Langmuir) binding model was used to fitthe data.

Epitope mapping of anti-DLL4 antibodies were done on Octet. AntiDLL-4antibodies C3. G6 and OMP21M18 were immobilized on AR2G sensor withamine-coupling kit, loaded rhDLL4, then ran different antibodies.Additional binding would indicate that the antibodies bind to differentepitope.

Results: This example illustrates an additional binding registered byOctet, indicating that C3, C5 and G6 antibodies bind to an epitope ofDLL-4 that is not occupied by OMP21M18.

Example 3

This example shows the blocking of DLL4 binding to Notch-1 by disclosedanti-DLL4-antibodies. Human DLL-4 is a type I transmembrane proteinexclusively expressed in endothelial cells. DLL-4 is a ligand for themembrane bound Notch-1 receptor. Upon binding to Notch-1, DLL-4activates Notch signaling pathway, which plays a major role inangiogenesis and tumor vascular development. Blocking of thisinteraction would inhibit Notch-1 function in cells. The blocking wasfirst evaluated in an ELISA-based immunoassay (FIG. 4A). Briefly, ratrecombinant Notch (5 μg/ml) was coated onto the wells of micro-titerplates at 4° C. overnight and the plate was then blocked with casein-PBSbuffer. His-tagged-rhDLL-4 (4 μg/ml) was pre-incubated with theanti-DLL-4 antibodies for 30 min, then transferred to the Notch-coatedplate and incubated for 0.5 hr with shaking. An HRP-conjugatedanti-his-tag antibody was used to detect binding of rhDLL-4 to Notch-1.

In parallel, the ability of anti-DLL-4 antibodies to block the bindingof soluble recombinant Notch-1 protein to DLL-4-expressing CHO-K1 cellswas assessed by HTFC. CHO-DLL-4 cells were lifted with enzyme-free CellDissociation Buffer (GIBCO), and incubated in FACS buffer+Azide with 20μg/ml FITC-rhNotch-1 in the presence or absence of anti-DLL-4 antibodiesat fixed (FIG. 4B) or increasing (FIG. 4C) concentrations. Data wereanalyzed by flow cytometry.

Results: FIG. 4A illustrates that 3 of the disclosed anti-DLL-4antibodies (C3, C10 and G6) were as efficient as OMP21M18 at blockingthe binding of DLL-4 to Notch-1. This assay was repeated in a cellularcontext (FIG. 4B), that confirms that C3, C10 and G6 are able toefficiently inhibit the interaction of human DLL-4 with both Human andRat Notch-1. These three antibodies appear more efficient (IC₅₀ valuesranging from 2 to 6 nM) than OMP21M18 (IC50=1.72 nM) (FIG. 4C).

Example 4

This example shows the effect of anti-DLL-4 antibodies on Notch-1pathway activation. Upon binding by DLL-4, the Notch-1 intracellulardomain (NICD) is cleaved and translocates to the nucleus to activate theexpression of specific target genes. Here we show that anti-DLL-4antibodies inhibit DLL-4-mediated Notch-1 pathway activation andmodulate expression of Notch target genes in a breast cancer cell Line.MCF7 cells expressing Notch-1 were co-incubated for 24 hr with CHO cellseither over-expressing Human DLL-4 (DLL-4) or not (No DLL-4). Cells werethen harvested and total RNA was extracted with Trizol (Life Technology)according to manufacturer's recommendations. Gene expression wasanalyzed by RT-PCR using Promega's Access RT-PCR System kit, accordingto the Company's instructions. The forward and reverse primers were asfollows: Notch-1 Forward CACTGTGGGCGGGTCC SEQ ID NO. 41, Notch-1Reverse: GTITGTATTGGTTCGGCACCAT SEQ ID NO. 42, HEY-1 Forward:GGAGAGGCGCCGCTGTAGTTA SEQ ID NO. 43, HEY-1 Reverse:CAAGGGCGTGCGCGTCAAAGTA, SEQ ID NO. 44 GAPDH Forward:GGACCTGACCTGCCGTCTAGAA, SEQ ID NO. 45 GAPDH Reverse:GGTGTCGCTGTTGAAGTCAGAG SEQ ID NO. 46. PCR samples were run on a 3%agarose gel and bands intensities were quantified using Image LabSoftware (BIORAD). Human GAPDH was used as a reference gene fornormalization.

Results: FIG. 5 shows that the presence of DLL-4 stimulated Hey-1 andNotch-1 expression, and that anti-DLL-4 antibody C3 efficientlyinhibited DLL-4 stimulation of Notch-1 target gene regulation.

Example 5

This example shows an in vitro cell proliferation assay illustrating theability of the disclosed anti-DLL-4 antibodies to antagonizeDLL-4-mediated growth inhibition of Human Umbilical Vein EndothelialCells (HUVEC). Angiogenesis is a complex finely regulated process. DLL-4inhibits VEGF-mediated endothelial cell proliferation, and is induced byVEGF as a negative feedback regulator, thus preventing overly extensiveangiogenic sprouting. It was previously shown that inhibition of DLL-4rendered endothelial cells hyperproliferative, causing defective cellfate specification or differentiation both in vitro and in vivo. Hence,blocking DLL-4 was shown to inhibit tumor growth in several tumormodels. In this assay, low passage HUVEC cells were obtained from LONZAand cultured in EGM™-2 media (LONZA). One day prior to experiment, 96well-plates were coated at 4° C. overnight with 0.1% Gelatin+2 μg/mlrHuDLL-4 (SinoBiologics) or 0.1% Bovine Serum Albumin (BSA, SIGMA). Thencells were lifted with StemPro Accutase (Life Technology) and added tothe plates at 5,000 cells/well in normal culture media, and returned for3 days incubation at 37° C. Results were analyzed using a standard MTTassay.

Results: FIG. 6 shows that HUVEC proliferation was inhibited when cellswere cultured on immobilized DLL-4, and that disclosed anti-DLL-4antibodies (C3, C10 and G6) were capable of restoring HUVECproliferation. This antagonism of DLL-4 function was as efficient, ormore potent than Oncomed's OMP21M18 antibody.

TABLE 1 Human Anti-D11-4 antibody sequences variable domain regionsClones Heavy chain Light chain C3 QVQLVQSGSELKKPGASVKVSCKASQSVLTQPPSASGTPGQRVTISCSGSSSNIG GYTFTSYYMHWVRQVPGQGLEWMGSNTVNWYQQFPGTAPKLLIYRNNQRPSGV IINPSGGSTSYAQKFQGRVTMTRDTSPDRFSGSKSGTSASLAISGLQSEDEADYY TSTVYMELSSLRSEDTAVYYCATDYYCAAWDDSPNGPVFGGGTKLTVL DSSGYVDFDYWGQGTLVTVSS SEQ ID No. 2 SEQ ID No. 1C5 QVQLQQSGPGLVKPSQTLSLTCAISG QSVVTQPPSVSAAPGQKVTISCSGGSSNIDSVSSNSAAWNWIRQSPSRGFEWL GNNYVSWYQQIPGTAPKLLIYDNNNRPSGIGRTEYRSKWYNDYAVSVESRITINPD PDRFSGSKSGASATLGISGLQTGDEANYYTSKNHFSLQLNSVTPEDTAVYYCARD CGAWDSTLSGYVFGTGTKLTVL GNTESEDYWGQGTLVTVSSSEQ ID NO. 4 SEQ ID NO. 3 C10 QVQLVESGAEVKKPGASVKVSCKASQSVLTQPASVSGSPGQSITISCTGTSSDVG GYTFTSYYMHWVRQAPQGLEWMGIIGYNYVSWYQQHPGKAPKLMIYDVTNRPS NPSGGSTSYAQKFQGRVTMTRDTSTGVSNRFSGSKSGNTASLTISGLQAEDEAD NTVYMELSSLRSEDTAVYYCARDVWYYCSSYTSSSTVIFGGGTKLTVL GGYFDYWGQGTLVTVSS SEQ ID NO. 6 SEQ ID NO. 5 G6EVQLVESGGSLVQPGGSLRLSCAAS QSVLTQPPSASGTPGQRVTISCSGSSSNIGGFTFSNGWMTWLRQAPGKGLEWVA SNTVNWYQQLPGTAPKLLIYSNNQRPSGVTIKPDGSDTAYVESVKGRFTISRDNA PDRFSGSKSGTSASLAISGLQSEDEADYYKNLLYLQMDSLRGDDTAVYFCARDL CAAWDGSLNGYVFGTGTKLTVL AYNAFDIWGQGTMVTVSSSEQ ID NO. 8 SEQ ID NO. 7 F11 EVQLVESGAEVKKPGASVKVSCKASSQPVLTQPASVSGSPGQSITISCTGTSSDI GYTFTSYYMHWVRQAPGQGLEWMGGDYNYVSWYQQHPGKAPKLMIYEVSKRPS IISPSGDSTSYAQKFQGRVTMTKDTSGVPDRFSGSKSGNTASLTISGLQAEDEAD TSTVSMELSSLRSEDTAVYYCARDQYYCYSYAGSYTYVFGTGTKLTV EGLRGSGYYGMDVWGQGTTVTVSS SEQ ID NO. 10SEQ ID NO. 9 D2 QVQLVESGAEVKKPGASVKVSCKAS QPVLTQPASVSGSPGQSITIPCTGTRSDVGGYTFTTYFIHWVRQAPGQGLEWMGII GYNFVSWYQQHPGKAPKLLIYDNNKRPSGNPSGGSTSYAQKFQGRVTMTRDTST IPGRFSGSKSGTSATLGITGLQTGDEADYYSTVYMELSSLRSEDTAVYYCAREFM CGTWDDSLNVWVFGGGTKVTVL ATGGFDYWGQGTLVTVSSSEQ ID NO. 12 SEQ ID NO. 11 E10 EVQLVQSGAEVKKPGASVKVSCKASAIRMTQSPSFLSASVGDRITITCRASQDISA GYSFMNYDVTWVRQAAQGLEWMGYLAWYQQKPGTAPKVLIYAASTLQSGVPS WMNPDSGNTGYADQFQGRITMTRDRFSGSGSGTEFTLTISSLQPEDFATYYCQQ TSKSTAYMELTSLRSDDTAVYFCARALYDYLPITFGPGTKVDIK EVEVPGYYYKYGMDVWGQGTTVTV SEQ ID NO. 14 SSSEQ ID NO. 13 F12 QVQLVQSGAEVKKPGESVIISCKSSGLPVLTQPRSVSGSPGQSVTISCTGTSSDV FTFTRTAIHWMRQAPGQSFEWVGWIGGYNYVSWYQHHPGKAPKLMIYDVTKRPS RGSNGDTSYSQKFRDKVTVTADTFSGVPDRFSGSKSGNTASLTISGLQAEDEAD STSYLALNRLTSEDTAVYYCAREHPTYYCSSYTSSSTPYVFGTGTKVTVL SWDPDFWGQGTLVTVSS SEQ ID NO. 16 SEQ ID NO. 15E5 QVQLQQSGPGLVQPSQTLSLTCVIS LPVLTQSPSASGTPGQRVTISCSGSTSNIGGDSVSNNNAAWTWIRQSPSRGLEW SNTVNWYQQFPGTAPKLLIYYNDQRPSGVLGRTYYRSQWYSDYAVSVKSRMTIN PDRFSGSKSGTSASLAISGLQSEDEADYYPDTSKNQFSLQLNSLTPEDTAVYYCA CAAWDDRLYGRLFGGGTKLIVLREEVMDHDAFDIWGPGTMVTVSS SEQ ID NO. 18 SEQ ID NO. 17 H2EVQLLESGGGLVQPGGSLRLSCAAS EIVMTQSPSSLSASVGDRVTITCRASQSISSGFNVSKNYMSWVRQAPGKGLEWVS YLNWYQQKPGKAPKLLIYAASSLQSGVPSSMSSSSSYKYYADSVKGRFTISRDNA RFSGSGSGTDFTLTISSLQPEDFATYYCQQKNSLYLQMNSLRAEDTAVYYCAREN SYSTPLTFGQGTRLEIK DREAFDIWGQGTMVTVSSSEQ ID NO. 20 SEQ ID NO. 19 A1 QVQLQQSGPGLVKPSQTLSLTCAISGQPVLTQPASVSGSPGQSITISCTGTSNDVG DSVSSNGVAWNWIRQSPSRGLEWLSYDLVSWYQQHPGKAPKLMTYDVSNRPS GRTYFNSKWYNDYAVSVESRITINPDGVSNRFSGAKSGNTASLTISGLQAEDEAD TSKNQFSLQLNSVTPEDTAVYYCARYYCSSYTTSSTVVFGGGTKLTVL GRVSAFDYWGQGTPVTVSS SEQ ID NO. 22 SEQ ID NO. 21A2 EVQLVQSGAEVKKPGASVKVSCKAS QPVLTQPASVSGSPGQSITISCTGTSSDVGGYTFTSDEINWVRQATGQGLEWLG GYNYVSWFQQHPGKAPKLMIYDVNNRPSWMNPHSGNTGYAQKFQGRVTMTRN GVSNRFSGSKSGNTASLTISGLQAEDGADTSISTADMELSSLTSDDTAVYYCARG YYCSSYTSSSTYVFGTGTKVTVLHYYESSGYFFYGMDVWGQGTTVTV SEQ ID NO. 24 SS SEQ ID NO. 23 A11QVQLQQSGPGLVKPSQTLSLTCAISG QSVLTQPPSVSEAPRQGVTISCSGSRSNIGDSVSSNSAAWNWIRQSPSRGLEWL NNPVSWYQQVPGKPPKLLIYFDDLLPSGVGRTYYRSKWYNDYAVSVKSRITINPD SDRFSASKSGTSASLAISGLQSDDEADYFCTSKNQFSLQLNSVTPEDTAVYYCARE AAWDDSLNGRVFGGGTKLTVL GDDYGDHFDYWGQGTLVTVSSSEQ ID NO. 26 SEQ ID NO. 25 C12 EVQLLESGAEVKKPGASVKVSCKASYVLTQPPSASGTPGQRVTISCSGSNSNIGS GYTFTSYAMHWVRQAPGQRLEWLGNAVNWYQHLPGTAPKLLIYSNNQRPSGVP HINAANGNTKYSQKFQGRVTITRDTSDRFSGSKSGTSASLAISGLQSEDESDYYCI ASTAYMELSSLRSEDTAVYYCARARAWDGSLSGYVFGTGTKVTVL SGSYLVDYWGQGTLVTVSS SEQ ID NO. 28 SEQ ID NO. 27 D9QVQLVESGAEVRKPGASVKVSCKAS QSVVTQPPSLSAAPGQRVSISCSGTSSNIGGYTFTDYAIHWVRQAPGQRLEWMG KNYVSWYQQVPGTAPRLLIYDNNKRASGIWINAGNGNTKYSQKFQGRVTITRDT PARFSGSKSATSATLDIAGLQTGDEADYFCSASTAYMELSSLRSEDAAVYYCARG ETWDSSLRAEIFGGGTKLTVL NGSGSYLVDYWGQGTLVTVSSSEQ ID NO. 30 SEQ ID NO. 29 E3 QVQLVESGAEVKKPGASVKVSCKASSYELMQPHSVSESPGKTVTISCTRSSGNIA GYTFTSYPMHWVRQAPGQRLEWMGSNYVRWYQQRRDSAPTVVIFDDDQKPSG WINAGNGDTKYSQKFQDRVTITSDTSVADRFSGSIDTSSNSASLTISGLNTDDEAA ASTAYMELSSLRSEDTAVYYCAKDYYYYCHSDESSTVIFGGRTKLTVL TSGTYQIDYWGQGTLVTVSS SEQ ID NO. 32 SEQ ID NO. 31E6 EVQLVQSGAEVKKPGASVKVSCKAS QSVVTQPPSVSAAPGQKVTISCSGSSSNIEGYTFTSYTMHWVRQAPGQRLEWMG NNYVSWYQQLPGTAPKLLIYDNNKRPSGIPWINAGNGNTKYSQKFQGRVTITRDTF DRFSGSKSGTSATLGITGLQTGDEADYYCASTAYMELSSLRSEDTAVYYCARSR GTWDSSLSAEVFGTGTKVTVL YNSGGSLVDYWGQGTLVTVSSSEQ ID NO. 34 SEQ ID NO. 33 F1 QVQLVQSGAEVKKPGTSVKVSCKTSQAGLTQPPSASGSPGQPVTISCSGTSGDV GFPFTTYFFHWARQAPGQRPEWMGGGYDYVSWYQQHPGKAPKLIIYDVNKRPS WIHGGNGNTKYSQKFQGRVTITRDTGVPDRFSGSKSGNTASLTVSGLQAEDEAD SASTAYMELSSLRSEDAAVYYCARGYHCSSYAGSNNVIFGGGTKLTVL NGSGSYLVDYWGQGTLVTVSS SEQ ID NO. 36SEQ ID NO. 35 F6 QVQLVESGAEVKKPGASVKVSCKASQPVLTQPPSVSVAPGNTASITCGENNIGSK GYTFTNFFMHWVRQAPGQGLEWMGSVHWYQQKPGQAPVLVIYYDSDRPSGIPE VINPSGPGTTYPQKFQDRVTMTRDTRFSGSNSGNTATLTISRVEAGDEADYYCQ STSTVYMELSSLRSDDTAVYYCARDLVWDSSSDSWVFGGGTKVTVL INSGWSGAFDIWGQGTMVTVSS SEQ ID NO. 38 SEQ ID NO. 37F10 QVQLVQSGAEVKEPGASVKVSCKTS QSVLTQPPSVSAAPGQKVTISCSGSSSNIAGFTFTQIYVHWVRQAPGQGLEWMG NSYVSWYQQLPGTAPKLLIYDNNQRPSGIPLVRPSGSSRIYGQNFQGRVTLTRDTS DRFSGSKSGTSATLGITGLQTGDEADYYCTSTVYMDLSSLTSEDTAVYYCVTDVA GTWDSSLSAGVFGTGTKLTVL GYGDGRVWGQGTLVTVSSSEQ ID NO. 40 SEQ ID NO. 39

We claim:
 1. A method for blocking an interaction between Notch-1 andDLL-4, comprising administering an effective amount of an antibody thatbinds to a Delta-like 4 (DLL-4) epitope, the antibody comprising: aheavy chain variable domain sequence comprising SEQ ID NO. 1, SEQ ID NO.5, or SEQ ID NO. 7; and a light chain variable domain sequencecomprising SEQ ID NO. 2, SEQ ID NO. 6, or SEQ ID NO.
 8. 2. The method ofclaim 1, wherein the antibody comprises a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 5/SEQ ID NO. 6, and SEQ ID NO. 7/SEQ IDNO.
 8. 3. The method of claim 1, wherein the antibody is selected fromthe group consisting of a Fab, a Fab′, a F(ab′)2, an Fv, a domainantibody (dAb), a single-chain antibody, a chimeric antibody, a diabody,a triabody, a tetrabody, a fully human antibody, a humanized antibody,and a chimeric antibody.
 4. The method of claim 3, wherein the antibodyis an IgG.
 5. A method for blocking an interaction between Notch-1 andDLL-4, comprising administering an effective amount of an antibody Fabfragment that binds to a DLL-4, epitope, the Fab fragment comprising: aheavy chain variable domain sequence comprising SEQ ID NO. 1, SEQ ID NO.5, or SEQ ID NO. 7, and a light chain variable domain sequencecomprising SEQ ID NO. 2, SEQ ID NO. 6, or SEQ ID NO.
 8. 6. The method ofclaim 5, wherein the Fab fragment comprises a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 5/SEQ ID NO. 6, and SEQ ID NO. 7/SEQ IDNO.
 8. 7. A method for blocking an interaction between Notch-1 andDLL-4, comprising administering an effective amount of a single-chainantibody, or fragment thereof, that binds to a DLL-4 epitope, thesingle-chain antibody comprising: a heavy chain variable domain sequencecomprising SEQ ID NO. 1, SEQ ID NO. 5, or SEQ ID NO. 7, and a lightchain variable domain sequence comprising SEQ ID NO. 2, SEQ ID NO. 6, orSEQ ID NO. 8, wherein the heavy chain variable domain and the lightchain variable domain are connected by a peptide linker.
 8. The methodof claim 7, wherein the single-chain antibody, or fragment thereof,comprises a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 5/SEQID NO. 6, and SEQ ID NO. 7/SEQ ID NO.
 8. 9. A method for blocking aninteraction between Notch-1 and DLL-4, comprising administering aneffective amount of an antibody that binds to a DLL-4 epitope, theantibody comprising: (a) a heavy chain variable domain comprising CDR1,CDR2 and CDR3 region amino acid sequences set forth in SEQ ID NO. 1, 5,or 7; and (b) a light chain variable domain comprising CDR1, CDR2 andCDR3 region amino acid sequences set forth in SEQ ID NO. 2, 6, or
 8. 10.The method of claim 9, wherein the antibody comprises: (a) a heavy chainvariable domain comprising CDR1, CDR2 and CDR3 region amino acidsequences set forth in SEQ ID NO. 1; and (b) a light chain variabledomain comprising CDR1, CDR2 and CDR3 region amino acid sequences setforth in SEQ ID NO.
 2. 11. The method of claim 9, wherein the antibodycomprises: (a) a heavy chain variable domain comprising CDR1, CDR2 andCDR3 region amino acid sequences set forth in SEQ ID NO. 5; and (b) alight chain variable domain comprising CDR1, CDR2 and CDR3 region aminoacid sequences set forth in SEQ ID NO.
 6. 12. The method of claim 9,wherein the antibody comprises: (a) a heavy chain variable domaincomprising CDR1, CDR2 and CDR3 region amino acid sequences set forth inSEQ ID NO. 7; and (b) a light chain variable domain comprising CDR1,CDR2 and CDR3 region amino acid sequences set forth in SEQ ID NO.
 8. 13.The method of claim 9, wherein the antibody is selected from the groupconsisting of a Fab, a Fab′, a F(ab′)2, an Fv, a domain antibody (dAb),a single-chain antibody, a chimeric antibody, a diabody, a triabody, atetrabody, a fully human antibody, a humanized antibody, and a chimericantibody.
 14. The method of claim 13, wherein the antibody is a Fab. 15.The method of claim 13, wherein the antibody is a single-chain antibody.16. The method of claim 13, wherein the antibody, wherein the antibodyis an IgG.
 17. A method for blocking an interaction between Notch-1 andDLL-4, comprising administering an effective amount of an antibody thatbinds to a DLL-4 epitope, the antibody comprising a heavy chain variabledomain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, 5, and 7, and further comprising a lightchain.
 18. The method of claim 17, wherein the heavy chain variabledomain comprises the amino acid sequence set forth in SEQ ID NO.
 1. 19.The method of claim 17, wherein the heavy chain variable domaincomprises the amino acid sequence set forth in SEQ ID NO.
 5. 20. Themethod of claim 17, wherein the heavy chain variable domain comprisesthe amino acid sequence set forth in SEQ ID NO.
 7. 21. A method forblocking an interaction between Notch-1 and DLL-4, comprisingadministering an effective amount of an antibody that binds to a DLL-4epitope, the antibody comprising a light chain variable domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO. 2, 6, and 8, and further comprising a heavy chain.
 22. Themethod of claim 21, wherein the light chain variable domain comprisesthe amino acid sequence set forth in SEQ ID NO.
 2. 23. The method ofclaim 21, wherein the light chain variable domain comprises the aminoacid sequence set forth in SEQ ID NO.
 6. 24. The method of claim 21,wherein the light chain variable domain comprises the amino acidsequence set forth in SEQ ID NO.
 8. 25. A method for blocking aninteraction between Notch-1 and DLL-4, comprising administering aneffective amount of an antibody that binds to a DLL-4 epitope, whereinthe antibody comprises a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 5/SEQ ID NO. 6, and SEQ ID NO. 7/SEQ ID NO.
 8. 26. Themethod of claim 25, wherein the the heavy chain/light chain variabledomain sequence is SEQ ID NO. 1/SEQ ID NO.
 2. 27. The method of claim25, wherein the the heavy chain/light chain variable domain sequence isSEQ ID NO. 5/SEQ ID NO.
 6. 28. The method of claim 25, wherein the theheavy chain/light chain variable domain sequence is SEQ ID NO. 7/SEQ IDNO. 8.